SIMULTANEOUS DETERMINATION OF METHYLURIC ACIDS IN BIOLOGICAL FLUIDS BY RP-HPLC ANALYSIS AFTER SOLID PHASE EXTRACTION

Abstract

An automated reversed phase high performance liquid chromatographic (RP-HPLC) method, using a multi-linear gradient elution, is described for the simultaneous analysis of five methyluric acids: 7-methyluric acid (7-MU), 1-methyluric acid (1-MU), 1,3-di-methyluric acid (1,3-DMU), 1,7-di-methyluric acid (1,7-DMU), and 1,3,7-tri-methyluric acid (1,3,7-TMU). The separation method was based on mobile-phase optimization and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. The analytical column used was octylsilica, MZ-Kromasil, 5 μm, 250 × 4 mm i.d. Elution was performed at ambient temperature with a mixture of acetate buffer (pH = 3.5) and methanol at a volume ratio 95:5, changing to 30:70 v/v, over a period of 15 min. Isocaffeine was used as internal standard at a concentration of about 8 ng/μL. Detection was performed with a variable wavelength UV-visible detector at 280 nm, resulting in detection limits of 0.1 ng per 10 μL injection, while linearity held up to 5 ng/μL for each compound. The statistical evaluation of the method was examined performing intra day (n=8) and inter day calibration (n=8) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries, ranging from 89% to 106%, were achieved using Oasis HLB cartridges with methanol as eluent, requiring small volumes, 40 mL of blood serum and 100 mL of urine.